Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Synergy is cooperative activity between a plurality of entities to produce an effect greater than from simple additivity. Powerful combination drug therapies that have become mainstays in clinical care were all developed from previously known single therapies, many of which have dose-limiting toxicities. The rationale for combination drug therapy is that by combining medicines, a lower dose of one or both may be given to achieve the desired response with fewer side effects, or more than a single drug is required to manage the disease, irrespective of side effects. Furthermore, combination drugs acting on distinct pathways may better overcome the drug resistance that develops more readily to single agent treatments (Nature Reviews Drug Discovery 3 (2004)). Combination therapies are the backbone of clinical care for HIV, cancer and some infectious diseases (Chou, T. C. (2006) Pharmacol. Rev. 58(3):621-81; Volberding et al. (2010) The Lancet, 376:49-62). Clinicians have recognized that single-modality drugs are ineffective for treating complex disease, especially when drug resistance mechanisms come into play. Discovering innovative combination treatments earlier in the pharmaceutical R&D continuum requires a radically new drug discovery approach to high throughput screening (HTS), which currently tests only single compounds, seeking only singly-acting drugs.
Systematic discovery of multi-component therapeutics is too labor intensive for routine deployment in drug screening. As early as 1928, Loewe (Erg Physiol. (1928) 27:47-187) observed and quantified effects of combinations of compounds that were different from, and not predicted by, the activities of the constituents. The concepts of synergy, additivity, and antagonism have been explored extensively, particularly in the fields of pharmacology and toxicology (Chou, T. C. (2006) Pharmacol. Rev. 58(3):621-81). Patients with infectious diseases and those with cancer have benefited from combination chemotherapy, in many cases the standard of care (Lane, D. (2006) Nature Biotech., 24:163-164). This clinical experience—that single agents alone are insufficient to treat many diseases—has led physicians to test combinations of drugs in patients as an explicit strategy for treatment improvement. This clinical mixing has generally been conducted with agents already known to be effective in the therapeutic area of interest, or where there is a clear scientific basis for the combination.
Borisy and co-workers (Lehar et al. (2009) Discov. Med., 8:185-90) extrapolated a bench-screening method from the powerful logic of clinical combination drug testing to detect synergistic responses. Their important studies show data strongly suggesting that synergistic interactions that may be attributable to the interconnected signaling networks existing within and between cells can be detected with surprisingly high frequency, but one has to look for them. Those studies used known drugs that were laboriously paired with each other and then tested as binary pools of two drugs each. The authors developed an approach they termed combinatorial-HTS (cHTS) to prepare known drugs or other known active compounds with the aid of common automated pipettors systematically creating every possible drug pair in all possible combinations to detect all possible synergistic pairings in a library of several hundred compounds. Their cHTS approach was the basis for forming a biotechnology startup called CombinatoRx (Boston, Mass.). The approach may be useful for detecting synergistic actions in known drugs in smaller sets of pharmacologically active compounds. However, the approach cannot be used practically to detect drug synergy in the large libraries of diverse compounds typically required for HTS because the method is simply too laborious. For example, a relatively small library of 100,000 compounds results in about 5 billion unique pairs (the formula for calculating the numbers of possible pairs from a library ‘n’ compounds is: (n−1)×(n/2)).
Conditioned screening (CS) is a recently described approach that seeks to detect new drugs that act in combination with a known drug. The CS method is the application of a screen run with and without a sensitizing amount of a known agent in order to identify new drugs that enhance the effect of the known agent (i.e. drug). This offers a powerful approach to identify new agents that are effective only in the presence of the known drug. When used to discover agents which kill under the sensitizing condition, this approach is termed a screen for a ‘synthetic lethal’ (Iglehart et al., (2009) N. Engl. J. Med., 361:189-191). Conditioned screening has been described as HTS for Synergy (HTSS) when used to discover agents that are synergistic to the actions of a drug to which resistance has developed. In the example described in published work (Zhang et al. (2007) Proc. Natl. Acad. Sci., 104:4606-11), a microbial natural product library of 20,000 extracts was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. A known drug, beauvericin, dramatically synergized KTC activity against diverse fungal pathogens as determined in a checkerboard assay.
Like cHTS described above, HTSS has been shown useful in standard bioassays to experimentally detect combination drugs that may have desirable synergistic actions. However, this approach also suffers from the limitation that only a single known drug is tested for synergy against a library of random compounds. It would be extremely useful to develop a method that could detect drug synergy in any bioassay, with any large chemical library—no matter how large—with far greater efficiency than methods hitherto described.